About one in five thousand babies are born lacking neurons in the lowest part of the intestine. This condition is known as Hirschsprung Disease (HSCR) and requires corrective surgery. The progenitors of the affected neurons are born adjacent to the developing spinal cord and subsequently migrate to populate the entire length of the digestive tract, forming the enteric nervous system (ENS). The neural precursors use a receptor tyrosine kinase, RET, to detect and migrate towards a chemoattractive ligand, Glial Cell Line-Derived Neurotrophic Factor (GDNF). Genetic analysis in humans and mice supports a central role for RET signaling in HSCR, yet the actual role in vivo is under debate. A major challenge for the ENS field is to identify the molecular signals required for ENS formation in vivo. We believe that using Drosophila, with its powerful genetics, we can uncover these signals. Preliminary data indicates that Drosophila Ret is required for ENS formation, and we have identified an additional, parallel signaling pathway. Specific aim #1 will confirm and extend these findings. There is a remarkable conservation of molecular function between invertebrates and humans, so we believe our results will be relevant to HSCR in humans. Straightforward genetic analysis will determine whether Ret and a novel unrelated pathway are required for migration, proliferation, differentiation or axon guidance of ENS cells in vivo (aim #1). This information will be relevant to HSCR, and to adult ENS conditions, such as seen in diabetes. We have identified a candidate co-receptor and ligand and are testing these in tissue culture for physical association and signaling potential (specific aim #2). This could create an opportunity to analyze GDNF signaling in a simple organism, which would have relevance to non-ENS conditions such as Parkinson's disease. We will also develop new reagents for studying Drosophila ENS formation based on Ret promoter analysis (specific aim #3). Preliminary evidence suggests that the parallel signaling pathway we have uncovered will be relevant to vertebrate ENS formation. Our research may offer novel therapeutic approaches to HSCR, including factors necessary for successful enteric neuron transplantation. PUBLIC HEALTH RELEVANCE: The Ret gene plays a central role formation of the enteric (gut) nervous system, being frequently mutated in Hirschsprung's Disease (HSCR). Despite the contribution of animal models to our understanding of HSCR, no invertebrate model has yet been established. This proposal aims to develop a Drosophila model of HSCR with the aim of elucidating the basic mechanisms of action of Ret and interacting genes.